Hepatocellular carcinoma (HCC) is the most prevalent cancer in human population and will lead to over 23,000 deaths in the United States this year alone. However, the molecular mechanisms driving hepatocellular carcinoma (HCC) growth and metastasis remain elusive, and there is a huge, unmet medical need to find new therapeutic targets. We performed an unbiased genome-wide RNA interference (RNAi) screen to identify factors that are necessary for the epigenetic silencing of a HCC tumor suppressor Hedgehog-Interacting Protein (HHIP). The RNAi screen was based on the rationale that factors necessary for epigenetic silencing of HHIP in HCC cells, could in principle promote HCC tumor growth. The RNAi screen identified three new drivers of HCC: B-cell lymphoma 6 (BCL6); histone deacetylase 9 (HDAC9); and CDC-like kinase 1 (CLK1). RNAi- based knockdown of these genes significantly inhibited HCC cell growth in culture and in a mouse model of HCC tumorigenesis. Remarkably, we found that these three genes are overexpressed in patient-derived samples of HCC. Furthermore, their overexpression correlates with liver fibrosis and microvascular invasion, both of which are markers of poor prognosis. For this Phase I project we have chosen to develop small molecule inhibitors of BCL6 because it is a novel therapeutic target currently not exploited for HCC. Our hypothesis is that pharmacological inhibition of BCL6 will suppress the HCC malignant phenotype. Aim 1 experiments will use two existing, but therapeutically limited, small molecule inhibitors to demonstrate the utility of this new HCC epigenetic therapeutic strategy using appropriate in vitro and in vivo models. The inhibitors will be tested for inhibition of HCC cell growth in soft-agar assays, a surrogate assay for mouse tumorigenesis. Cell-based assays will also determine whether BCL6 inhibitors modulate HCC cell HHIP expression and expression of other downstream effectors, such as cyclin D1, p53 and PIM1. A BCL6 inhibitor with efficacy in a mouse model of lymphoma will be tested for efficacy in an orthotopic xenograft-based mouse model of HCC tumorigenesis. Concurrently, in Aim 2, we will utilize our medicinal chemistry capabilities to create a focused library of up to 100 new candidate BCL6 small molecule inhibitors that retain the beneficial inhibitory pharmacophore features of two existing structurally distinct inhibitors but add or eliminate other chemical features to address their off-target effects and pharmacokinetic structural liabilities. Structural activity relationships for these new merged pharmacophore compounds will be assessed by inhibition of BCL6-SMRT binding and by their ability to block HCC cell growth in soft-agar assays. In Aim 3, we will proceed to in vivo efficacy testing of the top five candidates in a BCL6-dependent mouse xenograft model of HCC. A successful Phase I project will reveal new and effective HCC therapeutic strategies and provide novel BCL6 inhibitors for Phase II medicinal chemistry to optimize efficacy and drug-like properties. The long-term project goal is to develop a novel epigenetic therapy to significantly improve treatment of HCC patients.